A liposome based platelet substitute, the plateletsome, with hemostatic efficacy.

The complexity of platelet mediated hemostasis has hindered development of a platelet substitute for transfusion therapy. In the current study, the hemostatic efficacy of a liposome based modality, the plateletsome, is demonstrated. A deoxycholate extract of a platelet membrane fraction, with a minimum of 15 proteins including GPIb, GPIIb-IIIa and GPIV/III, was incorporated into sphingomyelin: phosphatidylcholine: monosialylganglioside or egg phosphatide small unilamellar vesicles by reverse-phase/sonication and French press extrusion. These plateletsomes decreased bleeding by 67% in the tail bleeding time in rats made thrombocytopenic (platelets < 30,000/microliters) with external irradiation (7-9Gy) by Cesium source. Efficacy was also demonstrated in the thrombocytopathic, Fawn-Hooded rat, but to a lesser extent than in the thrombocytopenic animals. Direct plateletsome infusion to the tail wound was more effective than systemic administration for all effective preparations. On post-mortem examination, no pathologic thrombi were detected by gross and histopathologic examination of the lungs, livers, kidneys, or spleens of thrombocytopenic or normal animals after plateletsome infusion. No evidence of intravascular coagulation, monitored by levels of circulating fibrinogen and platelet counts, was observed when plateletsomes were administered intravenously to rabbits. No deleterious effect, either inhibition or hyperaggregability, on platelet aggregation studies in vitro was observed. While further refinements are clearly required, this study indicates that liposomes bearing specific platelet proteins may provide a basis for a clinically applicable platelet substitute.

Effect of calcium channel blockers on platelet GPIIb-IIIa as a calcium channel in liposomes: comparison with effects on the intact platelet.

The platelet membrane glycoprotein IIb-IIIa complex is essential for platelet aggregation and functions as a fibrinogen receptor on the activated platelet. When incorporated into phospholipid vesicles, this glycoprotein complex can function as an apparent calcium channel which facilitates the transit of calcium across a phospholipid barrier. In order to further evaluate this calcium channel, the effect of calcium channel blockers of the dihydropyridine (nifedipine and nicardipine), arylalkylamine (verapamil) and benzothiazepine (diltiazem) classes were evaluated on GPIIb-IIIa liposomes with encapsulated fura-2 (a fluorescent calcium indicator). Nicardipine, verapamil, and nifedipine significantly inhibited calcium influx into GPIIb-IIIa liposomes; however, this required 190 microM, 400 microM, and 140 microM drug, respectively. These concentrations are 10-1,000 fold greater than those clinically obtainable. In contrast, diltiazem at concentrations greater than 220 microM and amiloride at concentrations greater than 800 microM showed no inhibitory effects. When aspirinized platelets were activated with 30 micrograms/ml bovine fibrillar collagen, both nicardipine and diltiazem produced a decrease in both the initial rise and maximum cytoplasmic calcium concentration. Parallel experiments were performed to assess the effects of verapamil, nicardipine, and diltiazem on platelet aggregation in platelet rich plasma. Nicardipine, 190-380 microM, induced a prolongation of the lag phase, but no effect on the final degree of platelet aggregation to collagen. Similar inhibition of platelet aggregation was seen with diltiazem and verapamil although the effect of diltiazem was less pronounced particularly at higher concentrations of collagen. No effect was seen on aggregation with 32 microM ADP which is release independent, or on the primary wave of low dose ADP induced platelet aggregation.(ABSTRACT TRUNCATED AT 250 WORDS)

Ligand inhibition of the platelet glycoprotein IIb-IIIa complex function as a calcium channel in liposomes.

Platelet glycoproteins IIb and IIIa function as a fibrinogen receptor on the activated platelet. We have shown that these glycoproteins can be incorporated onto the surface of phosphatidylcholine vesicles with retention of fibrinogen and antibody binding properties and can permit Ca2+ transit across the phospholipid bilayer. In the current study we demonstrate that this apparent Ca2+ channel function is specifically inhibited by the synthetic analogue of the fibrinogen gamma COOH-terminal peptide, His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val (His-12-Val), but not by the adhesive protein sequence Arg-Gly-Asp-Ser (RGDS). Prior incubation of IIb-IIIa liposomes with RGDS prevented Ca2+ transit inhibition by 25 microM His-12-Val, analogous to RGDS inhibition of His-12-Val binding to platelets. His-12-Val inhibited a minor component of transmembrane Ca2+ influx into ADP and thrombin-activated human platelets but had no effect on steady-state platelet 45Ca flux. These data indicate that ligand binding may exert a regulatory influence on transmembrane Ca2+ influx into activated platelets. The difference in inhibitory potency of the peptides studied may be related to differences in conformational changes in the glycoprotein IIb-IIIa complex induced by His-12-Val and RGDS, steric considerations, or differences in interactions with glycoprotein IIb Ca2+ binding domains.

Platelet glycoproteins IIb and IIIa as a calcium channel in liposomes.

Human platelet membrane glycoproteins IIb and IIIa (GPIIb and IIIa) were incorporated into phospholipid vesicles by the reverse-phase technique to assess the ability of GPIIb and IIIa to function as a Ca2+ channel. Movement of Ca2+ across the lipid bilayer was quantitated by injection of proteoliposomes with encapsulated Fura-2 into Ca2+ buffers and measurement of Fura-2 fluorescence as an indicator of Ca2+ influx. Reciprocally, to assess the function of proteins in an inside-out orientation, Ca2+-loaded vesicles were injected into Ca2+-free buffer and Ca2+ efflux monitored by a calcium electrode. Incorporation of the IIb-IIIa complex produced significant facilitation of Ca2+ movement across the lipid bilayer. No net transmembrane Ca2+ movement was seen with dissociated IIb and IIIa. Movement of Ca2+ was proportional to the transmembrane Ca2+ gradient. Ca2+ movement into the vesicles was inversely proportional to extravesicular NaCl from 25 to 150 mmol/L, analogous to several studies in the intact platelet. Adenosine triphosphate had no effect on Ca2+ movement into or out of the vesicles. Specific inhibition of a Ca2+ shift into the vesicles was seen with M148, a monoclonal antibody to IIb/IIIa, while no inhibition was observed with a panel of other anti-IIb/IIIa monoclonal antibodies. This suggests that a specific site on the complex or orientation of the complex is essential for calcium channel function. These data demonstrate that the GPIIb/IIIa complex can serve as a passive Ca2+ channel across a phospholipid bilayer and has the potential to play a role in Ca2+ flux across the platelet plasma membrane.